Hospital-Acquired Infections Project 1. Real time PCR for quantitation of cytomegalovirus (CMV) in peripheral blood of allogeneic hematopoietic stem cell transplant (HSCT) recipients.Administration of antiviral therapy for CMV based on evidence of reactivation has become the standard of care in management of HSCT recipients. The most commonly used test to detect CMV reactivation is currently immunoperoxidase staining of pp65 protein in peripheral blood neutrophils. This test, as done at NIH, requires microscopic examination of 400,000 neutrophils. A single neutrophil with pp65 protein is considered enough to begin therapy with ganciclovir, valganciclovir or foscarnet, all toxic and expensive drugs. Therapy is continued until the pp65 test, done weekly, has been negative for two weeks. Although it is unknown whether this strategy leads to excessive antiviral therapy, it is clear that the incidence of clinically detectable CMV disease is now uncommon. Limitations of the pp65 test in detecting reactivation are expensive technician time, inability to test after transplantation until the absolute neutrophil count has reached 1000/mcl, and an occasional patient who develops CMV disease before pp65 becomes positive. Real time PCR was employed to test the ability of quantitative estimates of CMV DNA in whole blood to measure CMV reactivation in allogeneic hematopoietic stem cell transplant recipients who were either CMV seropositive or transplanted from a seropositive donor. Assays were run by the Clinical Center Microbiology Laboratory under the direction of Dr. Steven Fischer. Samples were tested in pairs using a standard curve from 5 to 5000 copies per reaction. Each reaction represented the DNA extracted from 20 mcl of whole blood. Repeatability of values at or below 5 copies per reaction, which was the bottom standard, was assessed by retesting the same DNA from 65 specimens in which one of two paired reaction tubes gave copy numbers at or below 5. If both of the prior reactions were positive, repeat testing of paired samples gave a positive in at least one tube in 20 or 23 (87%). If one of the pairs was negative in the original run and other had 5 copies or less, then repeat testing found at least one tube positive in only 21 or 42 samples (50%). This result was interpreted to mean that repeatability of a positive test was good unless one of paired samples was negative and the other reaction was positive at only 5 copies or less. Fifty one patients were entered into this study and followed during the period between Jan. 4, 2000 and Feb. 12, 2002. CMV reactivation was defined as use of ganciclovir or foscarnet for treatment of CMV reactivation, based on either the presence of CMV antigen (pp65) in neutrophils or the clinical suspicion of disease. In this NIH-approved protocol, clinicians were not informed of the results, so that treatment was not based on PCR results. Patients were tested weekly during their first 100 days post transplant, the exceptions being patients who were discharged without followup or died. Additionally, pp65 was not determined until the patient?s white blood count had reached 1000/mcl. A total of 928 samples were assayed by PCR. On 831 samples, a pp65 antigen was performed within three days of the PCR, the exceptions occurring when neutropenia prevented determination of pp65 testing. Of the 51 patients, 33 had a total of 38 episodes of CMV reactivation. Defining a positive test as at least one copy of DNA in an equivalent of 40 mcl of whole blood, PCR detected reactivation in 34 of the 35 episodes that were also pp65 positive. Three patients were treated because of possible CMV disease but had neither positive PCR nor positive pp65 test. The one patient who was treated because of a positive pp65 but had a negative PCR was only pp65 positive on a single occasion with a single positive neutrophil. A positive PCR preceded a positive pp65 in 27 of 30 episodes, the PCR becoming positive a mean of 13.5 days before pp65. No patient developed clinical evidence of CMV disease at a time when the PCR was positive and the pp65 was either negative or could not be measured because of neutropenia. The PCR test stayed positive beyond the last positive pp65 in 15 episodes, the mean difference being 11 days. The PCR also remained positive after antiviral therapy was discontinued in 8 patients. Of these, two were considered to need retreatment 1 and 6 weeks later, respectively. False positive PCR tests were defined as occurrence of a positive test outside an episode, surrounded by at least two negative PCR tests on either side. No patient had two consecutive PCR tests outside an episode with two prior and two subsequent negative tests. False positive tests occurred in 10 PCR tests on 8 patients, constituting 1% of all tests performed and 15.6% of all patients studied.. The number of false positive PCR tests was decreased to 2 tests, one in each of 2 patients when a positive test was defined as both paired tubes positive and one tube containing more than 5 copies. With this more restrictive definition of positivity, no additional episodes of reactivation were undetected. False positive tests were rare with PCR. Calculation of false positive pp65 tests, for purposes of comparison, is fraught with the problem that all positive pp65 tests were treated, albeit sometimes with courses abbreviated because of low clinical suspicion. In summary, quantitative PCR was able to detect CMV reactivation prior to pp65 in 27 of 30 episodes detected by pp65 (90%). None of the patients in this study developed clinical evidence of CMV disease at a time when PCR was positive and pp65 was negative. Persistence of PCR positivity beyond the end of antiviral therapy did not necessarily predict recurrence of CMV reactivation. Real time PCR can be used to replace pp65 antigenemia testing but clinical advantage was not detected during the first 100 days post transplant in a population of largely nonmyeloablative transplant recipients. Project 2. Prophylaxis of respiratory syncytial virus (RSV) pneumonia in HSCT recipients using of immune globulin (RSVIG) Occurrence of five RSV pneumonia cases, four of whom died, in our HSCT unit over a six week period (August through September, 1998) prompted us to begin a surveillance system to monitor the incidence of respiratory viral infections, including RSV, in our inpatient and outpatient HSCT patients. We also began an IRB-approved protocol with an open study of RSVIG immunoprophylaxis. Based on use of RSVIG in animals and infants, RSVIG seemed likely to prevent RSV upper respiratory infection from progressing to pneumonia, while not decreasing the number of RSV upper respiratory infections. We anticipated enrolling 120 patients over two years. With an incidence of RSV infection of 15% during the first 6 weeks and 60% progressing to pneumonia, a reduction from the anticipated 11 cases of pneumonia to a single case would be significant at the 5% level. After 54 patients had been screened and 32 patients followed for 42 days, we determined that only 4 patients who had received RSVIG were shown to have RSV infection and none with pneumonia. Of the 31 who chose not to enroll, 4 had RSV infection, none with pneumonia. It was determined that the incidence of RSV infection was currently too low to complete the study and the high cost of RSVIG was no longer warranted. The study was discontinued and the serologic response to RSVIG analyzed, with the results as follows. Thirty-two recipients of an allogeneic HSCT were given RSVIG at the time of transplantation and again three weeks later. Antibody titers to RSV, human parainfluenza virus 3 (HPIV3), measles and influenza H1N1, H3N2 and B were measured prior to RSVIG and five times subsequently over the next six weeks. Baseline antiviral titers and increase in antibody from RSVIG administration were extremely heterogeneous for all the viruses. All of the 32 patients had RSV antibody titers prior to RSVIG in the range considered protective for neonates. Titers that might protect immunosuppressed adults are not known. Eighteen patients had a baseline titer to RSV F protein at the lower end of spectrum among our patients (1:640-1:2048). These 18 had a 7.7 fold initial rise in titer following the first dose of RSVIG compared to only a 2.1 fold rise in 14 patients with higher baseline titers (1:4096-20,840). Increase in antibody titers against the other viruses following RSVIG infusion also reflected similar heterogeneity. The subset of patients with the lowest titers appears to stand the greatest benefit from RSVIG administration.